Compositions and Methods for Detecting Vitamin D

ABSTRACT

Compounds include carbamate derivatives of vitamin D including vitamin D 3  and vitamin D 2 . The compounds are useful in methods and kits for determining the presence and/or amount of vitamin D including vitamin D analogs and metabolites thereof in a sample suspected of containing the same.

BACKGROUND

This invention relates to compositions, methods and kits for determiningthe presence and/or amount of vitamin D including vitamin D analogs andmetabolites thereof in a sample suspected of containing the same.

The term “vitamin D” refers to a group of fat-soluble secosteroids. Inhumans, vitamin D is unique because it can be ingested ascholecalciferol (vitamin D₃) or ergocalciferol (vitamin D₂) and becausethe body can also synthesize it (from cholesterol) when sun exposure isadequate. Because of this latter property, vitamin D is considered bysome to be a non-essential dietary vitamin although most consider it anessential nutrient. Vitamin D has an important physiological role in thepositive regulation of calcium ion homeostasis. Vitamin D₃ is the formof the vitamin synthesized by animals. It is also a common supplementadded to milk products and certain food products as is vitamin D₂.

Both dietary and intrinsically synthesized vitamin D₃ must undergometabolic activation to generate bioactive metabolites. In humans, theinitial step of vitamin D₃ activation occurs primarily in the liver andinvolves hydroxylation to form the intermediate metabolite25-hydroxycholecalciferol (also referred to as calcidiol, calcifediol,25-hydroxycholecalciferol, or 25-hydroxyvitamin D (abbreviated 25(OH)D).The latter compound, i.e., 25-hydroxyvitamin D, is the specific vitaminD metabolite that is measured in serum to determine vitamin D status.Calcidiol is the major form of Vitamin D₃ in the circulatory system.Circulating calcidiol is converted by the kidney to1,25-dihydroxyvitamin D₃ (calcitriol; 1,25(OH)₂D₃), which is believed tobe the most biologically active form of vitamin D.

Assessing vitamin D levels in biological samples is important sincevitamin D deficiency is related to a number of disorders in mammals.There is a need for reagents and methods for accurate and sensitivedeterminations of concentrations of vitamin D, vitamin D analogs andmetabolites thereof in samples.

SUMMARY

Some examples in accordance with the principles described herein aredirected to a compound of the formula:

wherein:

Z is an alkyl, alkenyl or alkynyl group having 1 to 10 carbon atoms,which may be unsubstituted or one or more of which may be substituted byone or more of hydroxy, lower alkoxy, oxo and oxime;

R is —O-succinimidyl, —NH—O-(A)_(k)-R², wherein R² is a member of asignal producing system, —(CH₂)_(n)COOH, —(CH₂)_(m)COONHS (NHS isN-hydroxysuccinimide), a small molecule, a binding partner for a smallmolecule, or a support; A is a linking group; k is 0 or 1; n is aninteger of 1 to 5 and m is an integer of 1 to 5; and

R⁴ is H, OH or lower alkoxy.

Some examples in accordance with the principles described herein aredirected to a compound of the formula:

or a compound of the formula:

wherein:

R′ is —O-succinimidyl or —NH—O-(A)_(k)-R², wherein R² is a member of asignal producing system, —(CH₂)_(n)COOH, —(CH₂)_(m)COONHS (NHS isN-hydroxysuccinimide), a small molecule, a binding partner for a smallmolecule, or a support; A is a linking group; k is 0 or 1; n is aninteger of 1 to 5 and m is an integer of 1 to 5;

R¹ is H, OH, or protected OH; and

R⁴ is H, OH or lower alkoxy.

Some examples in accordance with the principles described herein aredirected to a compound of the formula:

or a compound of the formula:

wherein:L is selected from the group consisting of chemiluminescent particlesand sensitizer particles and R^(1′) is OH and A′ is a linking group.

Some examples in accordance with the principles described herein aredirected to methods of determining one or both of the presence and theamount of vitamin D in a sample suspected of containing vitamin D. Inthe method a combination in a medium is provided that comprises thesample, a compound as described above and a specific binding member forvitamin D. The combination is subjected to conditions for binding of thecompound to the specific binding member to form a complex. The amount ofthe complex is measured where the amount of the complex is related toone or both of the presence and amount of vitamin D in the sample.

Some examples in accordance with the principles described herein aredirected to methods of determining one or both of the presence and theamount of vitamin D in a sample suspected of containing vitamin D. Acombination is provided that comprises the sample, a specific bindingmember for vitamin D and a compound of the formula:

or a compound of the formula:

wherein:R″ is —NH—O-A″—R^(2′), wherein R^(2′) is a member of a signal producingsystem and A″ is a linking group, and

R^(1″) is H or OH.

The combination is subjected to conditions for binding of the compoundto the specific binding member to form a complex, and the amount of thecomplex is measured. The amount of the complex is related to thepresence and/or amount of vitamin D in the sample.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic diagram of a synthesis of compounds in accordancewith examples in accordance with the principles described herein.

FIG. 2 is a schematic diagram of a synthesis of compounds in accordancewith examples in accordance with the principles described herein.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS Compounds

As mentioned above, some examples in accordance with the principlesdescribed herein are directed to compounds of the formula:

wherein:

Z is an alkyl, alkenyl or alkynyl group having, for example, 1 to 10, or1 to 9, or 1 to 8, or 1 to 7, or 1 to 6, or 1 to 5, or 1 to 4, or 1 to3, or 1 to 2, or 2 to 10, or 2 to 9, or 2 to 8, or 2 to 7, or 2 to 6, or2 to 5, or 2 to 4, or 2 to 3, or 3 to 10, or 3 to 9, or 3 to 8, or 3 to7, or 3 to 6, or 3 to 5, or 3 to 4, or 4 to 10, or 4 to 9, or 4 to 8, or4 to 7, or 4 to 6, or 4 to 5, or 5 to 10, or 5 to 9, or 5 to 8, or 5 to7, or 5 to 6, or 6 to 10, or 6 to 9, or 6 to 8, or 6 to 7, or 7 to 10,or 7 to 9, or 7 to 8, or 8 to 10, or 8 to 9, or 9 to 10, carbon atoms,which may be unsubstituted or one or more of which may be substituted byone or more of hydroxy, alkoxy of 1 to 5, or 1 to 4, of 1 to 3, or 1 to2, or 2 to 5, or 2 to 4, or 2 to 3, or 3 to 5, or 3 to 4, or 4 to 5carbon atoms, oxo, or oxime; and

R is —O-succinimidyl or —NH—O-(A)_(k)-R², R² is a member of a signalproducing system, —(CH₂)_(n)COOH, —(CH₂)_(m)COONHS (NHS isN-hydroxysuccinimide), a small molecule, a binding partner for a smallmolecule, or a support; A is a linking group; k is 0 or 1; n is aninteger of 1 to 5, or 1 to 4, of 1 to 3, or 1 to 2, or 2 to 5, or 2 to4, or 2 to 3, or 3 to 5, or 3 to 4, or 4 to 5 and m is an integer of 1to 5, or 1 to 4, of 1 to 3, or 1 to 2, or 2 to 5, or 2 to 4, or 2 to 3,or 3 to 5, or 3 to 4, or 4 to 5; and

R⁴ is H, OH or lower alkoxy.

The term “alkyl” includes those alkyl groups of a designated number ofcarbon atoms of either a straight, branched, or cyclic configuration.Examples of “alkyl” include, but are not limited to, methyl, ethyl,propyl, isopropyl, butyl, sec- and tert-butyl, pentyl, hexyl, heptyl,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,norbornyl, for example.

The term “alkenyl” includes hydrocarbon chains of a specified number ofcarbon atoms of either a straight- or branched-configuration and atleast one carbon-carbon double bond, which may occur at any point alongthe chain, examples of which include, but are not limited to, ethenyl,propenyl, butenyl, pentenyl, dimethyl pentenyl, for example.

The term “alkynyl” refers to a straight or branched chain hydrocarbon ofa specified number of carbon atoms containing at least one carbon-carbontriple bond, including, but not limited to, ethynyl, 1-propynyl,2-propynyl, 1-butynyl, and 2-butynyl, for example.

The term “alkoxy” includes alkyl groups of a designated number of carbonatoms of either a straight, branched or cyclic configuration wherein thealkyl group includes an ether oxygen for linking to a parent compound.

In some examples in accordance with the principles described herein, thelinking group A has a molecular weight less than about 2000, or lessthan about 1500, or less than about 1000, or less than about 500, orless than about 300, or less than about 200, or less than about 150, forexample. The linking group may comprise about 2 to about 200 atoms, or 4to about 150 atoms, or about 5 to about 100 atoms, or about 5 to about50 atoms, or about 5 to about 25 atoms, not counting hydrogen, and maycomprise a chain of from 2 to about 100 atoms, or 3 to about 90 atoms,or about 4 to about 80 atoms, or about 5 to about 70 atoms, or about 10to about 50 atoms, or about 10 to about 25 atoms, or about 5 to about 20atoms, or about 5 to about 10 atoms, for example, each independentlyselected from the group consisting of carbon, oxygen, sulfur, nitrogen,and phosphorous. The number of heteroatoms in the linking group isdependent on the size of the linking group and, in some examples, thenumber is in the range of from 0 to about 30, or 1 to about 25, or about2 to about 20, or about 2 to about 15, or about 2 to about 10, or about3 to about 10, or about 3 to about 5, for example.

The heteroatoms may be in the form of one or more functionalitiesincluding, but not limited to, one or more of amine (primary, secondaryor tertiary), carbamate, ether, ester, amide, urea, sulfonamide,thioether, hydrazone, hydrazide, amidine, and phosphate ester, forexample. In one example in accordance with the principles describedherein, the linking group comprises two nitrogen atoms in the form ofsecondary amine functionalities and one carbonyl group in a chain ofabout 6 to about 20, or about 6 to about 15, or about 6 to about 10, orabout 7 to about 20, or about 7 to about 15, or about 7 to about 10, orabout 7 to about 9, or about 7 to about 8, or about 7 atoms, whichnumber of atoms includes carbon atoms.

For the most part, when a linking group has a linking functionality(functionality for reaction with a moiety) such as, for example, anon-oxocarbonyl group including nitrogen and sulfur analogs, a phosphategroup, an amino group, alkylating agent such as halo or tosylalkyl, oxy(hydroxyl or the sulfur analog, mercapto) oxocarbonyl (e.g., aldehyde orketone), or active olefin such as a vinyl sulfone or α-,β-unsaturatedester, these functionalities are linked to amine groups, carboxylgroups, active olefins, alkylating agents, e.g., bromoacetyl. Where anamine and carboxylic acid or its nitrogen derivative or phosphoric acidare linked, amides, amidines and phosphoramides are formed. Wheremercaptan and activated olefin are linked, thioethers are formed. Wherea mercaptan and an alkylating agent are linked, thioethers are formed.Where aldehyde and an amine are linked under reducing conditions, analkylamine is formed. Where a ketone or aldehyde and a hydroxylamine(including derivatives thereof where a substituent is in place of thehydrogen of the hydroxyl group) are linked, an oxime functionality(═N—O—) is formed. Where a carboxylic acid or phosphate acid and analcohol are linked, esters are formed.

As mentioned above, in some examples in accordance with the principlesdescribed herein, R² may be a member of a signal producing system. Thesignal producing system may have one or more components, at least onecomponent being a label. The signal producing system generates a signalthat relates to the presence of vitamin D in a sample. The signalproducing system includes all of the reagents required to produce ameasurable signal. Other components of the signal producing system maybe included in a developer solution and can include, but are not limitedto, substrates, enhancers, activators, chemiluminescent compounds,cofactors, inhibitors, scavengers, metal ions, and specific bindingsubstances required for binding of signal generating substances, forexample. Other components of the signal producing system may becoenzymes, substances that react with enzymic products, other enzymesand catalysts, for example. The signal producing system provides asignal detectable by external means, by use of electromagneticradiation, desirably by visual examination. Exemplary signal-producingsystems are described in U.S. Pat. No. 5,508,178, the relevantdisclosure of which is incorporated herein by reference.

The term “label” includes poly(amino acid) labels and non-poly(aminoacid) labels. The term “poly(amino acid) label moieties” includes labelsthat are proteins such as, but not limited to, enzymes, antibodies,peptides, and immunogens, for example. With label proteins such as, forexample, enzymes, the molecular weight range will be from about 10,000to about 600,000, or from about 10,000 to about 300,000 molecularweight. There is usually at least one compound in accordance with theprinciples described herein (analog group) per about 200,000 molecularweight, or at least about 1 per about 150,000 molecular weight, or atleast about 1 per about 100,000 molecular weight, or at least about 1per about 50,000 molecular weight, for example, of the protein. In thecase of enzymes, the number of analog groups is usually from 1 to about20, about 2 to about 15, about 3 to about 12, or about 6 to about 10.

Enzymes include, by way of illustration and not limitation, redoxenzymes such as, for example, dehydrogenases, e.g., glucose-6-phosphatedehydrogenase and lactate dehydrogenase; enzymes that involve theproduction of hydrogen peroxide and the use of the hydrogen peroxide tooxidize a dye precursor to a dye such as, for example, horseradishperoxidase, lactoperoxidase and microperoxidase; hydrolases such as, forexample, alkaline phosphatase and β-galactosidase; luciferases such as,for example firefly luciferase, and bacterial luciferase; transferases;combinations of enzymes such as, but not limited to, saccharideoxidases, e.g., glucose and galactose oxidase, or heterocyclic oxidases,such as uricase and xanthine oxidase, coupled with an enzyme thatemploys hydrogen peroxide to oxidize a dye precursor, that is, aperoxidase such as horseradish peroxidase, lactoperoxidase ormicroperoxidase, for example.

The term “non-poly(amino acid) labels” includes those labels that arenot proteins. The non-poly(amino acid) label is capable of beingdetected directly or is detectable through a reaction that produces adetectable signal. The non-poly(amino acid) label can be isotopic ornon-isotopic and can be, by way of illustration and not limitation, aradioisotope, a luminescent compound (which includes, but is not limitedto fluorescent compounds and chemiluminescent compounds, for example), apolynucleotide coding for a catalyst, a promoter, a dye, a coenzyme, anenzyme substrate, a radioactive group, and an amplifiable polynucleotidesequence, for example.

As mentioned above, in some examples in accordance with the principlesdescribed herein, R² may be a small organic molecule refers to amolecule of molecular weight of about 200 to about 2,000, or about 200to about 1,500, or about 200 to about 1,000, or about 200 to about 500.Such small organic molecules include, but are not limited to, biotin,fluorescent molecules (such as fluorescein and rhodamine, for example),chemiluminescent molecules and dinitrophenol, for example. A bindingpartner for a small organic molecule is a molecule that specificallyrecognizes and binds to the small molecule. Binding partners for a smallmolecule are defined by the nature of the small molecule and include,but are not limited to, avidin, streptavidin, antibody for the smallorganic molecule (which include, but are not limited to, antibody for afluorescent molecule (such as antibody for fluorescein and antibody forrhodamine, for example), antibody for a chemiluminescent molecule,antibody for dinitrophenol, for example.

As mentioned above, in some examples in accordance with the principlesdescribed herein, R² may be a support, which may be comprised of anorganic or inorganic, solid or fluid, water insoluble material and whichmay be transparent or partially transparent. The support can have any ofa number of shapes, such as, but not limited to, a particle (particulatesupport) including bead, a film, a membrane, a tube, a well, a strip, arod, a fiber, or a planar surface such as, e.g., a plate or paper, forexample. The support may or may not be suspendable in the medium inwhich it is employed. Examples of suspendable supports are polymericmaterials such as latex, lipid bilayers or liposomes, oil droplets,cells and hydrogels, and magnetic particles, for example. Other supportcompositions include polymers, such as, by way of illustration and notlimitation, nitrocellulose, cellulose acetate, poly (vinyl chloride),polyacrylamide, polyacrylate, polyethylene, polypropylene, poly (4methylbutene), polystyrene, polymethacrylate, poly(ethyleneterephthalate), nylon, poly(vinyl butyrate), for example, either used bythemselves or in conjunction with other materials. The support may ormay not be further labeled with a dye, catalyst or other detectablegroup, for example.

In some examples in accordance with the principles described herein, thesupport may be a particle. The particles have an average diameter of atleast about 0.02 microns and not more than about 100 microns. In someexamples, the particles have an average diameter from about 0.05 micronsto about 20 microns, or from about 0.3 microns to about 10 microns. Theparticle may be organic or inorganic, swellable or non-swellable, porousor non-porous, preferably of a density approximating water, generallyfrom about 0.7 g/mL to about 1.5 g/mL, and composed of material that canbe transparent, partially transparent, or opaque. The particles can bebiological materials such as cells and microorganisms, e.g.,erythrocytes, leukocytes, lymphocytes, hybridomas, streptococcus,Staphylococcus aureus, and E. coli, viruses, for example. The particlescan also be particles comprised of organic and inorganic polymers,liposomes, latex particles, magnetic or non-magnetic particles,phospholipid vesicles, chylomicrons, lipoproteins, and the like. In someexamples, the particles are chromium dioxide (chrome) particles or latexparticles.

In some examples in accordance with the principles described herein, Zin the above formula is —(CH₂)_(s)C(CH₃)₂—R¹ wherein s is an integer of1 to 5, or 1 to 4, of 1 to 3, or 1 to 2, or 2 to 5, or 2 to 4, or 2 to3, or 3 to 5, or 3 to 4, or 4 to 5, or 3, or 2, or 1, and R¹ is H, OH,or protected OH. In a particular example R¹ is OH. In a particularexample in accordance with the principles described herein, s is 3 andthe compound has the formula:

wherein:

R′ is —O-succinimidyl or —NH—O-(A)_(k)-R², wherein R² is a member of asignal producing system, —(CH₂)_(n)COOH, —(CH₂)_(m)COONHS (NHS isN-hydroxysuccinimide), a small molecule, or a binding partner for asmall molecule, a support, A is a linking group, k is 0 or 1, n is aninteger of 1 to 5 and m is an integer of 1 to 5;

R¹ is H, OH, or protected OH. In a particular example, R¹ is OH; and

R⁴ is H, OH or lower alkoxy. In a particular example, R⁴ is H. Inanother particular example, R⁴ is OH.

In some examples in accordance with the principles described herein, Zin the above formula is —(CH═CH)(CH₂)_(v)CH(CH₃)(CH₃)₂—R¹ wherein v isan integer of 0 to 5, or 0 to 4, of 0 to 3, or 0 to 2, or 0 to 1, or 1to 5, or 1 to 4, of 1 to 3, or 1 to 2, or 2 to 5, or 2 to 4, or 2 to 3,or 3 to 5, or 3 to 4, or 4 to 5, or 3, or 2, or 1, and R¹ is H, OH, orprotected OH. In a particular example R¹ is OH. In a particular examplein accordance with the principles described herein, v is 0 and thecompound has the formula:

wherein:R′ is —O-succinimidyl or —NH—O-(A)_(k)-R², wherein R² is a member of asignal producing system, —(CH₂)_(n)COOH, —(CH₂)_(m)COONHS (NHS isN-hydroxysuccinimide), a small molecule, or a binding partner for asmall molecule, a support, A is a linking group, k is 0 or 1, n is aninteger of 1 to 5 and m is an integer of 1 to 5;

R¹ is H, OH, or protected OH. In a particular example, R¹ is OH; and

R⁴ is H, OH or lower alkoxy. In a particular example, R⁴ is H. Inanother particular example, R⁴ is OH.

Some examples in accordance with the principles described herein aredirected to derivatives of 25-OH vitamin D₂ and derivatives of 25-OHvitamin D₃ that comprise a carbamate linkage to another moiety eitherdirectly or through the intermediacy of a linking group. The moiety maybe, but is not limited to, a member of a signal producing system, asupport or a member of a specific binding pair.

In some examples in accordance with the principles described herein, thecompound has the formula:

wherein:

L is selected from the group consisting of chemiluminescent particlesand sensitizer particles and R^(1′) is OH and A′ is a linking group asdescribed above. In some examples, the linking group has the formula—(CH₂)_(p)—C(O)—(W)_(b)—(CH₂)_(q)—(X)_(c)—(CH₂)_(r)— wherein W and X areeach independently —NH— and —O—, b and c are each independently 0 or 1,p is an integer of 1 to 5, or 1 to 4, or 1 to 3, or 1 to 2, or 2 to 5,or 2 to 4, or 2 to 3, or 3 to 5, or 3 to 4, or 4 to 5, q is an integerof 1 to 5, or 1 to 4, or 1 to 3, or 1 to 2, or 2 to 5, or 2 to 4, or 2to 3, or 3 to 5, or 3 to 4, or 4 to 5 and r is an integer of 1 to 5, or1 to 4, or 1 to 3, or 1 to 2, or 2 to 5, or 2 to 4, or 2 to 3, or 3 to5, or 3 to 4, or 4 to 5. In one example, b and c are each 1, p is 1, qis 2 and r is 1. In some examples, the linking group has the formula—(CH₂)_(p)—C(O)—NH—(CH₂)_(q)—NH—(CH₂)_(r)— wherein p, q and r are asdefined above.

In some examples in accordance with the principles described herein, thecompound has the formula:

wherein:

L is selected from the group consisting of chemiluminescent particlesand sensitizer particles and R^(1′) is OH and A′ is a linking group asdescribed above. In some examples, the linking group has the formula—(CH₂)_(p)—C(O)—(W)_(b)—(CH₂)_(q)—(X)_(c)—(CH₂)_(r)— wherein W and X areeach independently —NH— and —O—, b and c are each independently 0 or 1,p is an integer of 1 to 5, or 1 to 4, or 1 to 3, or 1 to 2, or 2 to 5,or 2 to 4, or 2 to 3, or 3 to 5, or 3 to 4, or 4 to 5, q is an integerof 1 to 5, or 1 to 4, or 1 to 3, or 1 to 2, or 2 to 5, or 2 to 4, or 2to 3, or 3 to 5, or 3 to 4, or 4 to 5 and r is an integer of 1 to 5, or1 to 4, or 1 to 3, or 1 to 2, or 2 to 5, or 2 to 4, or 2 to 3, or 3 to5, or 3 to 4, or 4 to 5. In one example, b and c are each 1, p is 1, qis 2 and r is 1. In some examples, the linking group has the formula—(CH₂)_(p)—C(O)—NH—(CH₂)_(q)—NH—(CH₂)_(r)— wherein p, q and r are asdefined above.

Chemiluminescent particles are particles that have associated therewitha chemiluminescent compound. The phrase “associated therewith” as usedherein means that a compound such as, for example, a chemiluminescentcompound and a particle may be associated by direct or indirect bonding,adsorption, absorption, incorporation, or solution, for example.Examples of chemiluminescent compounds that may be utilized are thoseset forth in U.S. Pat. Nos. 5,340,716 and 6,251,581, the relevantdisclosures of which are incorporated herein by reference. In someexamples in accordance with the principles described herein, thechemiluminescent compound is a photoactivatable substance that undergoesa chemical reaction upon direct or sensitized excitation by light orupon reaction with singlet oxygen to form a metastable reaction productthat is capable of decomposition with the simultaneous or subsequentemission of light, usually within the wavelength range of 250 to 1200nm. The term “photoactivatable” includes “photochemically activatable”.In some examples, the chemiluminescent compounds are those that reactwith singlet oxygen to form dioxetanes or dioxetanones. The latter areusually electron rich olefins. Exemplary of such electron rich olefinsare enol ethers, enamines, 9-alkylidene-N-alkylacridans,arylvinylethers, dioxenes, arylimidazoles, 9-alkylidene-xanthanes andlucigenin. Other compounds include luminol and other phthalhydrazidesand chemiluminescent compounds that are protected from undergoing achemiluminescent reaction by virtue of their being protected by aphotochemically labile protecting group, such compounds including, forexample, firefly luciferin, aquaphorin, and luminol. Examples of suchchemiluminescent compounds that may be utilized are those set forth inU.S. Pat. No. 5,709,994, the relevant disclosure of which isincorporated herein by reference.

Sensitizer particles are particles that have associated therewith asensitizer compound, which includes, but is not limited to, aphotosensitizer compound. Examples of sensitizer compounds that may beutilized are those set forth in U.S. Pat. Nos. 5,340,716 and 6,251,581,the relevant disclosures of which are incorporated herein by reference.

A photosensitizer is a sensitizer for generation of singlet oxygenusually by excitation with light. In some examples, the photosensitizerabsorbs at a longer wavelength than the chemiluminescent compound andhas a lower energy triplet than the chemiluminescent compound. Thephotosensitizer can be photoactivatable (e.g., dyes and aromaticcompounds). The photosensitizer is usually a compound comprised ofcovalently bonded atoms, usually with multiple conjugated double ortriple bonds. The compound should absorb light in the wavelength rangeof 200-1100 nm, usually 300-1000 nm, preferably 450-950 nm. Typicalphotosensitizers include, but are not limited to, acetone, benzophenone,9-thioxanthone, eosin, 9,10-dibromoanthracene, methylene blue,metallo-porphyrins (e.g., hematoporphyrin), phthalocyanines,chlorophylls, rose bengal, buckminsterfullerene, for example, andderivatives of these compounds. Examples of other photosensitizers areenumerated in N. J. Turro, “Molecular Photochemistry”, page 132, W. A.Benjamin Inc., N.Y. 1965. The photosensitizer assists photoactivationwhere activation is by singlet oxygen. Usually, the photosensitizerabsorbs light and the thus formed excited photosensitizer activatesoxygen to produce singlet oxygen, which reacts with the chemiluminescentcompound to give a metastable luminescent intermediate.

In some examples in accordance with the principles described herein, thecompound has the formula:

wherein L, p, q and r are as defined above.

In some examples in accordance with the principles described herein, thecompound has the formula:

wherein L, p, q and r are as defined above.

Preparation of Compounds

Examples of methods of preparing compounds in accordance with theprinciples described herein are described, by way of illustration andnot limitation, with reference to FIGS. 1 and 2. Other approaches may beemployed to form the compounds consistent with the principles describedherein. Referring to FIG. 1, 25-OH VD₃ (I) is treated to formsuccinimidyl carbonate derivative II. For example, I may be combinedwith disuccinimidyl carbonate in the presence of a base in an anhydrouspolar organic solvent. The base may be, for example, triethylamine ordiisopropylethylamine. The anhydrous polar organic solvent may be, forexample, acetonitrile, dichloromethane, tetrahydrofuran (THF),dimethylformamide (DMF) or dimethylsulfoxide (DMSO). The temperatureduring the reaction is about 18° C. to about 25° C., or about roomtemperature. The reactants are subjected to agitation during thereaction by stirring or shaking, for example. The time period of thereaction is about 2 hours to about 24 hours, or about 15 to about 21hours, or about 18 hours.

Carbamate III (25-OH vitamin D₃ 3-carbamate, p=1) may be prepared fromsuccinimidyl carbonate derivative II by, for example, combining II witha suspension of carboxymethoxylamine hemihydrochloride (CMO) in thepresence of a base in an anhydrous polar organic solvent. The base maybe, for example, triethylamine or diisopropylethylamine. The anhydrouspolar organic solvent may be, for example, DMF or DMSO. The reactionvessel containing the above reactants is held in a heating bath at aheating bath temperature during the reaction of about 18° C. to about25° C., or about 22° C. The reactants are subjected to agitation duringthe reaction by stirring or shaking, for example. The time period of thereaction is about 15 to about 21 hours, or about 18 hours. The organicsolvent is removed by evaporation, which may be accelerated bysubjecting the contents of the reaction vessel to a vacuum. The residuemay be combined with a polar organic solvent such as, for example, ethylacetate or dichloromethane and then washed with an aqueous solution ofan inorganic salt such as, for example, brine or sodium bicarbonatesolution. The organic phase may be dried by, for example, mixing with ananhydrous inorganic salt such as, sodium sulfate or magnesium sulfate,for example. Following drying, the organic phase may be treated toseparate any solid material by subjecting the organic phase to, forexample, filtration or decantation. The organic solvent is removed byevaporation, which may be accelerated by subjecting the contents of thereaction vessel to a vacuum. The resulting dry product may be purifiedby chromatographic means such as, for example, high performance liquidchromatography (HPLC), reverse phase liquid chromatography (RPLC), highturbulence liquid chromatography (HTLC), or gas chromatography. Theproduct may be stored in an anhydrous polar organic solvent such as, forexample, DMSO or DMF.

Carbamate III may be activated for coupling to a linking group of asolid support such as, for example, a particle. In one example inaccordance with the principles described herein, carbamate III isactivated by formation of sulfo-NHS ester IV. The reaction is carriedout by combining carbamate III with an activation agent such as, forexample, sulfo-NHS, in the presence of, for example,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) in an anhydrouspolar organic medium such as, for example, DMSO, at a temperature ofabout 15° C. to about 40° C., or about 20° C. to about 30° C., or atambient temperature for a period of about 10 hours to about 25 hours, orabout 12 hours to about 20 hours, or about 18 hours. The medium issubjected to agitation such as by rotating or stirring, for example.

Referring to FIG. 2, a particle such as, for example, a latex particle(designated EPRM in this example) having incorporated therein achemiluminescent compound (dye) and having an inner aminodextran layerand an outer dextran aldehyde layer is prepared by a procedure similarto that described in U.S. Pat. No. 7,179,660, the relevant disclosure ofwhich is incorporated herein by reference. Aldehyde groups on the outerdextran aldehyde layer are reacted with ethylene diamine under reductiveamination conditions to form reagent V (EPRM-EDA), which is a particlereagent having pendant moieties comprising an ethylene chain and aterminal amine group. The reductive amination conditions include the useof a reducing agent such as, for example, a metal hydride (e.g., sodiumborohydride or potassium borohydride), sodium cyanoborohydride, orsodium triacetoxyborohydride. The reaction is carried out in an aqueousmedium at a temperature during the reaction of about 20° C. to about100° C., or about 30° C. to about 50° C., for a period of about 1 hourto about 48 hours, or about 5 hours to about 24 hours. The point oflinkage of the pendant moieties with the outer dextran aldehyde layercomprises a secondary amine linkage.

Sulfo-NHS ester IV (in an anhydrous polar organic solvent such as, forexample, DMSO or DMF) is combined with particle reagent V (in ananhydrous polar organic solvent such as, for example, DMSO or DMF, witha surfactant such as, for example, GAFAC® or TWEEN® 20). The reactionmixture is subjected to agitation by, for example, stirring or shaking.The temperature during the reaction is about 18° C. to about 25° C., orabout room temperature. The reactants are subjected to agitation duringthe reaction by stirring or shaking, for example. The time period of thereaction is about 2 hours to about 24 hours, or about 15 to about 21,for example. The resulting particle (VI) may be subjected to one or morewashing and purification techniques such as, for example, diafiltrationand sonication, for example.

Vitamin D₂ derivatives in accordance with the principles describedherein may be prepared in a manner similar to that described above forthe preparation of vitamin D₃ derivatives in accordance with theprinciples described herein.

General Description of Assays for Vitamin D Utilizing the PresentCompounds

Some examples in accordance with the principles described herein aredirected to methods of determining one or both of the presence and theamount of vitamin D in a sample suspected of containing vitamin D andmay be referred to herein as “assays for vitamin D.” In the assay acombination in a medium is provided that comprises the sample, acompound as described above comprising a member of a signal producingsystem and a specific binding member for vitamin D. As used herein inreference to assays, the term “vitamin D” refers to one or more of25-hydroxycholecalciferol (also referred to as calcidiol, calcifediol,25-hydroxycholecalciferol, or 25-hydroxyvitamin D (abbreviated 25(OH)D);calcidiol; 1,25-dihydroxyvitamin D₃ (calcitriol; 1,25(OH)₂D₃);1,25-dihydroxy vitamin D₄; 1,25-dihydroxy vitamin D₅; and 1,25-dihydroxyvitamin D₆; including metabolites of all of the above.

One particular example in accordance with the principles describedherein is directed to methods of determining one or both of the presenceand the amount of vitamin D in a sample suspected of containing vitaminD. A combination is provided that comprises the sample, a specificbinding member for vitamin D and a compound of the formula:

or a compound of the formula:

wherein: R″ is —NH—O-A″—R^(2′), wherein R² is a member of a signalproducing system as defined above and A″ is a linking group as definedabove, and R″ is H or OH. The combination is subjected to conditions forbinding of the compound to the specific binding member to form acomplex, and the amount of the complex is measured. The amount of thecomplex is related to the presence and/or amount of vitamin D in thesample.

The specific binding member is a member of a specific binding pair,which is one of two different molecules, having an area on the surfaceor in a cavity, which specifically binds to and is thereby defined ascomplementary with a particular spatial and polar organization of theother molecule. The members of the specific binding pair will usually bemembers of an immunological pair such as antigen-antibody, althoughother specific binding pairs such as biotin-avidin, hormones-hormonereceptors, enzyme-substrate, nucleic acid duplexes, IgG-protein A,polynucleotide pairs such as DNA-DNA, DNA-RNA, for example, are notimmunological pairs but are included within the scope of the term “sbpmember.”

Specific binding involves the specific recognition of one of twodifferent molecules for the other compared to substantially lessrecognition of other molecules. On the other hand, non-specific bindinginvolves non-covalent binding between molecules that is relativelyindependent of specific surface structures. Non-specific binding mayresult from several factors including hydrophobic interactions betweenmolecules. Preferred binding partners are antibodies.

In some examples of assays in accordance with the principles describedherein, the specific binding member for vitamin D is an antibody forvitamin D, which may be a complete immunoglobulin molecule or a fragmentthereof. Antibodies include various classes and isotypes, such as IgA,IgD, IgE, IgG1, IgG2a, IgG2b and IgG3, and IgM, for example. Fragmentsthereof may include Fab, Fv and F(ab′)₂, Fab′, and the like. Inaddition, aggregates, polymers, and conjugates of immunoglobulins ortheir fragments can be used where appropriate so long as bindingaffinity for vitamin D is retained. Antibodies for vitamin D may beprepared by techniques including, but not limited to, immunization of ahost and collection of sera (polyclonal), preparing continuous hybridcell lines and collecting the secreted protein (monoclonal) or cloningand expressing nucleotide sequences or mutagenized versions thereofcoding at least for the amino acid sequences required for specificbinding of natural antibodies, for example.

The sample to be analyzed is one that is suspected of containing vitaminD. The samples may be biological samples or non-biological samples.Biological samples may be from a mammalian subject or a non-mammaliansubject. Mammalian subjects may be, e.g., humans or other animalspecies. Biological samples include biological fluids such as wholeblood, serum, plasma, sputum, lymphatic fluid, semen, vaginal mucus,feces, urine, spinal fluid, saliva, stool, cerebral spinal fluid, tears,mucus, and the like; biological tissue such as hair, skin, sections orexcised tissues from organs or other body parts; and so forth. In manyinstances, the sample is whole blood, plasma or serum. Non-biologicalsamples including, but not limited to, waste streams, for example, mayalso be analyzed using compounds in accordance with the principlesdescribed herein.

The sample can be prepared in any convenient medium, which may be, forexample, an assay medium, which is discussed more fully hereinbelow. Insome instances a pretreatment may be applied to the sample such as, forexample, to lyse blood cells. In some examples, such pretreatment isperformed in a medium that does not interfere subsequently with anassay.

The combination in the medium is subjected to conditions for binding ofthe compound to the specific binding member for vitamin D to form acomplex. The amount of the complex is measured where the amount of thecomplex is related to one or both of the presence and amount of vitaminD in the sample.

An assay for vitamin D can be performed either without separation(homogeneous) or with separation (heterogeneous) of any of the assaycomponents or products. Heterogeneous assays usually involve one or moreseparation steps and can be competitive or non-competitive. Immunoassaysmay involve labeled or non-labeled reagents. Immunoassays involvingnon-labeled reagents usually comprise the formation of relatively largecomplexes involving one or more antibodies prepared from immunogenicconjugates in accordance with the principles described herein. Suchassays include, for example, immunoprecipitin and agglutination methodsand corresponding light scattering techniques such as, e.g.,nephelometry and turbidimetry, for the detection of antibody complexes.Labeled immunoassays include, but are not limited to, chemiluminescenceimmunoassays, enzyme immunoassays, fluorescence polarizationimmunoassays, radioimmunoassays, inhibition assays, induced luminescenceassays, and fluorescent oxygen channeling assays, for example.

One general group of immunoassays includes immunoassays using a limitedconcentration of a compound in accordance with the principles describedherein. Another group of immunoassays involves the use of an excess ofone or more of the principal reagents such as, for example, an excess ofa compound in accordance with the principles described herein. Anothergroup of immunoassays includes separation-free homogeneous assays inwhich a labeled reagent in accordance with the principles describedherein modulates the label signal upon binding of a compound inaccordance with the principles described herein to a specific bindingmember for vitamin D, thus competing with vitamin D that may be presentin the sample.

As mentioned above, the assays can be performed either withoutseparation (homogeneous) or with separation (heterogeneous) of any ofthe assay components or products. Homogeneous immunoassays areexemplified by the EMIT® assay (Siemens Healthcare Diagnostics Inc.,Deerfield, Ill.) disclosed in Rubenstein, et al., U.S. Pat. No.3,817,837, column 3, line 6 to column 6, line 64; immunofluorescencemethods such as those disclosed in Ullman, et al., U.S. Pat. No.3,996,345, column 17, line 59, to column 23, line 25; enzyme channelingimmunoassays (“ECIA”) such as those disclosed in Maggio, et al., U.S.Pat. No. 4,233,402, column 6, line 25 to column 9, line 63; thefluorescence polarization immunoassay (“FPIA”) as disclosed, forexample, in, among others, U.S. Pat. No. 5,354,693; and enzymeimmunoassays such as the enzyme linked immunosorbant assay (“ELISA”).Exemplary of heterogeneous assays are the radioimmunoassay, disclosed inYalow, et al., J. Clin. Invest. 39:1157 (1960). The relevant portions ofthe above disclosures are all incorporated herein by reference.

Other enzyme immunoassays are the enzyme modulate mediated immunoassay(“EMMIA”) discussed by Ngo and Lenhoff, FEBS Lett. (1980) 116:285-288;the substrate labeled fluorescence immunoassay (“SLFIA”) disclosed byOellerich, J. Clin. Chem. Clin. Biochem. (1984) 22:895-904; the combinedenzyme donor immunoassays (“CEDIA”) disclosed by Khanna, et al., Clin.Chem. Acta (1989) 185:231-240; homogeneous particle labeled immunoassayssuch as particle enhanced turbidimetric inhibition immunoassays(“PETINIA”), particle enhanced turbidimetric immunoassay (“PETIA”),etc.; and the like.

Other assays include the sol particle immunoassay (“SPIA”), the dispersedye immunoassay (“DIA”); the metalloimmunoassay (“MIA”); the enzymemembrane immunoassays (“EMIA”); luminoimmunoassays (“LIA”); and soforth. Other types of assays include immunosensor assays involving themonitoring of the changes in the optical, acoustic and electricalproperties of the present conjugate upon the binding of vitamin Danalyte. Such assays include, for example, optical immunosensor assays,acoustic immunosensor assays, semiconductor immunosensor assays,electrochemical transducer immunosensor assays, potentiometricimmunosensor assays, amperometric electrode assays.

Heterogeneous assays usually involve one or more separation steps andcan be competitive or non-competitive. A variety of competitive andnon-competitive heterogeneous assay formats are disclosed in Davalian,et al., U.S. Pat. No. 5,089,390, column 14, line 25 to column 15, line9, incorporated herein by reference. In an example of a competitiveheterogeneous assay, a support having an antibody for vitamin D boundthereto is contacted with a medium containing the sample suspected ofcontaining vitamin D and a labeled compound in accordance with theprinciples described herein. Vitamin D in the sample competes, forbinding to the vitamin D antibody, with the compound in accordance withthe principles described herein bearing a detectable label. Afterseparating the support and the medium, the label activity of the supportor the medium is determined by conventional techniques and is related tothe amount of vitamin D analyte in the sample.

In some examples, a sample to be analyzed is combined in an assay mediumwith an antibody for vitamin D and labeled compound in accordance withthe principles described herein. The medium is examined for one or bothof the presence and amount of a complex comprising the labeled compoundand the antibody for vitamin D where the presence and/or the amount ofsuch complex indicates the presence and/or amount of vitamin D in thesample.

The assays are normally carried out in an aqueous buffered medium at amoderate pH, generally that which provides optimum assay sensitivity.The aqueous medium may be solely water or may include from 0.1 to about40 volume percent of a cosolvent. The pH for the medium will be in therange of about 4 to about 11, or in the range of about 5 to about 10, orin the range of about 6.5 to about 9.5, for example. The pH will usuallybe a compromise between optimum binding of the binding members of anyspecific binding pairs, the pH optimum for other reagents of the assaysuch as members of the signal producing system, and so forth. Variousbuffers may be used to achieve the desired pH and maintain the pH duringthe assay. Illustrative buffers include, by way of illustration and notlimitation, borate, phosphate, carbonate, tris, barbital, PIPES, HEPES,MES, ACES, MOPS, and BICINE, for example. The particular buffer employedis not critical, but in an individual assay one or another buffer may bepreferred.

Various ancillary materials may be employed in the assay methods. Forexample, in addition to buffers the medium may comprise stabilizers forthe medium and for the reagents employed. In some embodiments, inaddition to these additives, proteins may be included, such as, forexample, albumins; organic solvents such as, for example, formamide;quaternary ammonium salts; polyanions such as, for example, dextransulfate; binding enhancers, for example, polyalkylene glycols;polysaccharides such as, for example, dextran or trehalose. The mediummay also comprise agents for preventing the formation of blood clots.Such agents are well known in the art and include, but are not limitedto, EDTA, EGTA, citrate, heparin, for example. The medium may alsocomprise one or more preservatives such as, but not limited to, sodiumazide, neomycin sulfate, PROCLIN® 300, Streptomycin, for example. Themedium may additionally comprise one or more surfactants. Any of theabove materials, if employed, is present in a concentration or amountsufficient to achieve the desired effect or function.

One or more incubation periods may be applied to the medium at one ormore intervals including any intervals between additions of variousreagents employed in an assay including those mentioned above. Themedium is usually incubated at a temperature and for a time sufficientfor binding of various components of the reagents and binding of vitaminD in the sample to occur. Moderate temperatures are normally employedfor carrying out the method and usually constant temperature,preferably, room temperature, during the period of the measurement. Insome examples, incubation temperatures range from about 5° to about 99°C., or from about 15° C. to about 70° C., or from about 20° C. to about45° C., for example. The time period for the incubation, in someexamples, is about 0.2 seconds to about 24 hours, or about 1 second toabout 6 hours, or about 2 seconds to about 1 hour, or about 1 minute toabout 15 minutes, for example. The time period depends on thetemperature of the medium and the rate of binding of the variousreagents, which is determined by the association rate constant, theconcentration, the binding constant and dissociation rate constant.

In an example of a method for determining vitamin D in a samplesuspected of containing vitamin D, a combination is provided in a mediumwhere the combination includes the sample, an antibody for vitamin D,and a labeled compound in accordance with the principles describedherein where the label is a poly(amino acid) label or a non-poly(aminoacid) label. The medium is examined for one or both of the presence andamount of one or both of a complex comprising vitamin D and the antibodyfor vitamin D or a complex comprising the labeled compound and antibodyfor vitamin D. The presence and/or the amount of one or both of thecomplexes indicates the presence and/or amount of vitamin D in thesample.

Some known assays utilize a signal producing system (sps) that employsfirst and second sps members. The designation “first” and “second” iscompletely arbitrary and is not meant to suggest any order or rankingamong the sps members or any order of addition of the sps members in thepresent methods. The sps members may be related in that activation ofone member of the sps produces a product such as, e.g., light or anactivated product, which results in activation of another member of thesps.

In some embodiments of assays, the sps members comprise a sensitizersuch as, for example, a photosensitizer, and a chemiluminescentcomposition where activation of the sensitizer results in a product thatactivates the chemiluminescent composition. The second sps memberusually generates a detectable signal that relates to the amount ofbound and/or unbound sps member, i.e., the amount of sps member bound ornot bound to the vitamin D analyte being detected or to a compound inaccordance with the principles described herein. In some examples inaccordance with the principles described herein, one of either thesensitizer reagent or the chemiluminescent reagent comprises the presentcompound reagent.

In a particular example, an induced luminescence immunoassay may beemployed. The induced luminescence immunoassay is referred to in U.S.Pat. No. 5,340,716 (Ullman), which disclosure is incorporated herein byreference. In one approach, the assay uses a particle having associatedtherewith a photosensitizer where a compound in accordance with theprinciples described herein is bound to the particle (particle-compoundreagent). The chemiluminescent reagent comprises an antibody for vitaminD. The vitamin D analyte competes with the particle-compound reagent forbinding to the antibody for vitamin D. If the vitamin D analyte ispresent, the fewer is the number of molecules of particle-compoundreagent that come into close proximity with the chemiluminescentreagent. Therefore, there will be a decrease in the assay signal. Thephotosensitizer generates singlet oxygen and activates thechemiluminescent reagent when the two labels are in close proximity. Theactivated chemiluminescent reagent subsequently produces light. Theamount of light produced is related to the amount of the complex formed,which in turn is related to the amount of vitamin D analyte present inthe sample.

In another particular example of an induced luminescence immunoassay,the assay uses a particle having associated therewith a chemiluminescentcompound where a compound in accordance with the principles describedherein is bound to the particle (particle-compound reagent). Thephotosensitizer reagent comprises an antibody for vitamin D. The vitaminD analyte competes with the particle-compound reagent for binding to theantibody for vitamin D. If the vitamin D analyte is present, the feweris the number of molecules of particle-compound reagent that come intoclose proximity with the photosensitizer reagent. Therefore, there willbe a decrease in the assay signal. The photosensitizer generates singletoxygen and activates the chemiluminescent compound of theparticle-compound reagent when the two labels are in close proximity.The activated chemiluminescent compound subsequently produces light. Theamount of light produced is related to the amount of the complex formed,which in turn is related to the amount of vitamin D analyte present inthe sample.

In another particular example of an induced luminescence assay, aphotosensitizer particle is employed that is conjugated to a bindingpartner for a small molecule such as, for example, avidin orstreptavidin (which are binding partners for biotin). A compound inaccordance with the principles described herein that comprises biotin(compound-biotin reagent) is also employed. A chemiluminescent reagentthat comprises a specific binding member for vitamin D is employed aspart of the detection system. The reaction medium is incubated to allowthe avidin or streptavidin of the photosensitizer particles to bind tothe compound-biotin reagent by virtue of the binding between avidin andbiotin and to also allow the specific binding member for the vitamin Dthat is part of the chemiluminescent reagent to bind to the vitamin Danalyte or to the compound in accordance with the principles describedherein that is now attached to the photosensitizer particles. Then, themedium is irradiated with light to excite the photosensitizer, which iscapable in its excited state of activating oxygen to a singlet state.Because less of the chemiluminescent reagent is now in close proximityto the photosensitizer because of the presence of the vitamin D analyte,there is less activation of the chemiluminescent reagent by the singletoxygen and less luminescence. The medium is then examined for thepresence and/or the amount of luminescence or light emitted, thepresence thereof being related to the presence and/or amount of thevitamin D analyte where a decrease in signal is observed in the presenceof the vitamin D analyte.

In another particular example of an induced luminescence assay, aphotosensitizer particle is employed that is conjugated to a bindingpartner for a small molecule such as, for example, avidin orstreptavidin (which are binding partners for biotin). A conjugatereagent comprises a specific binding member for vitamin D conjugated tobiotin. A compound in accordance with the principles described herein isemployed where the compound is attached to a chemiluminescent particle(chemiluminescent-compound reagent) is also employed. The reactionmedium is incubated to allow the avidin or streptavidin of thephotosensitizer particles to bind to the antibody-biotin reagent byvirtue of the binding between avidin and biotin and to also allow thespecific binding member for the vitamin D to bind to vitamin D ifpresent in the sample and to the compound in accordance with theprinciples described herein that is part of thechemiluminescent-compound reagent. Then, the medium is irradiated withlight to excite the photosensitizer, which is capable in its excitedstate of activating oxygen to a singlet state. Because less of thechemiluminescent-compound reagent is now in close proximity to thephotosensitizer because of the presence of the vitamin D analyte, thereis less activation of the chemiluminescent reagent by the singlet oxygenand less luminescence. The medium is then examined for the presenceand/or the amount of luminescence or light emitted, the presence thereofbeing related to the presence and/or amount of the vitamin D analytewhere a decrease in signal is observed in the presence of the vitamin Danalyte.

The concentration of the vitamin D analyte in a sample that may beassayed generally varies from about 10⁻⁵ to about 10⁻¹⁷ M, or from about10⁻⁶ to about 10⁻¹⁴ M, for example. Considerations such as whether theassay is qualitative, semi-quantitative or quantitative (relative to theamount of the vitamin D analyte present in the sample), the particulardetection technique and the expected concentration of the vitamin Danalyte normally determine the concentrations of the various reagents.

The concentrations of the various reagents in the assay medium willgenerally be determined by the concentration range of interest of thevitamin D analyte, the nature of the assay, and the like. However, thefinal concentration of each of the reagents is normally determinedempirically to optimize the sensitivity of the assay over the range ofinterest. That is, a variation in concentration of vitamin D analytethat is of significance should provide an accurately measurable signaldifference. Considerations such as the nature of the signal producingsystem and the nature of the analytes normally determine theconcentrations of the various reagents.

As mentioned above, the sample and reagents are provided in combinationin the medium. While the order of addition to the medium may be varied,there will be certain preferences for some embodiments of the assayformats described herein. The simplest order of addition, of course, isto add all the materials simultaneously and determine the effect thatthe assay medium has on the signal as in a homogeneous assay.Alternatively, each of the reagents, or groups of reagents, can becombined sequentially. In some embodiments, an incubation step may beinvolved subsequent to each addition as discussed above. Inheterogeneous assays, washing steps may also be employed after one ormore incubation steps.

Examination Step

In a next step of an assay method, the medium is examined for thepresence of a complex comprising the vitamin D analyte and antibody forvitamin D and/or a complex comprising a compound reagent in accordancewith the principles described herein and antibody for vitamin D. Thepresence and/or amount of one or both of the complexes indicates thepresence and/or amount of the vitamin D analyte in the sample.

The phrase “measuring the amount of a vitamin D analyte” refers to thequantitative, semiquantitative and qualitative determination of vitaminD. Methods that are quantitative, semiquantitative and qualitative, aswell as all other methods for determining the vitamin D analyte, areconsidered to be methods of measuring the amount of the vitamin Danalyte. For example, a method, which merely detects the presence orabsence of the vitamin D analyte in a sample suspected of containing thevitamin D analyte, is considered to be included within the scope of thepresent invention. The terms “detecting” and “determining,” as well asother common synonyms for measuring, are contemplated within the scopeof the present invention.

In many embodiments the examination of the medium involves detection ofa signal from the medium. The presence and/or amount of the signal isrelated to the presence and/or amount of the vitamin D analyte in thesample. The particular mode of detection depends on the nature of thesignal producing system. As discussed above, there are numerous methodsby which a label of a signal producing signal can produce a signaldetectable by external means. Activation of a signal producing systemdepends on the nature of the signal producing system members.

Temperatures during measurements generally range from about 10° C. toabout 70° C. or from about 20° C. to about 45° C., or about 20° C. toabout 25° C., for example. In one approach standard curves are formedusing known concentrations of vitamin D analyte. Calibrators and othercontrols may also be used.

Luminescence or light produced from any label can be measured visually,photographically, actinometrically, spectrophotometrically, such as byusing a photomultiplier or a photodiode, or by any other convenientmeans to determine the amount thereof, which is related to the amount ofvitamin D analyte in the medium. The examination for presence and/oramount of the signal also includes the detection of the signal, which isgenerally merely a step in which the signal is read. The signal isnormally read using an instrument, the nature of which depends on thenature of the signal. The instrument may be, but is not limited to, aspectrophotometer, fluorometer, absorption spectrometer, luminometer,and chemiluminometer, for example.

Kits Comprising Reagents for Conducting Assays

A reagent comprising a compound in accordance with the principlesdescribed herein attached to a member of a signal producing system,which may also comprise a support, and other reagents for conducting aparticular assay for a vitamin D analyte may be present in a kit usefulfor conveniently performing an assay for the determination of a vitaminD analyte. In some embodiments a kit comprises in packaged combination abiotin-binding partner such as, for example, avidin or streptavidin,associated with a particle, biotinylated compound in accordance with theprinciples described herein and a labeled antibody for the vitamin Danalyte. The kit may further include other reagents for performing theassay, the nature of which depend upon the particular assay format.

The reagents may each be in separate containers or various reagents canbe combined in one or more containers depending on the cross-reactivityand stability of the reagents. The kit can further include otherseparately packaged reagents for conducting an assay such as additionalspecific binding pair members, signal producing system members, andancillary reagents, for example.

The relative amounts of the various reagents in the kits can be variedwidely to provide for concentrations of the reagents that substantiallyoptimize the reactions that need to occur during the present methods andfurther to optimize substantially the sensitivity of an assay. Underappropriate circumstances one or more of the reagents in the kit can beprovided as a dry powder, usually lyophilized, including excipients,which on dissolution will provide for a reagent solution having theappropriate concentrations for performing a method or assay using acompound reagent in accordance with the principles described herein. Thekit can further include a written description of a method utilizingreagents that include a compound reagent in accordance with theprinciples described herein.

The phrase “at least” as used herein means that the number of specifieditems may be equal to or greater than the number recited. The phrase“about” as used herein means that the number recited may differ by plusor minus 10%; for example, “about 5” means a range of 4.5 to 5.5.

The following discussion is directed to specific examples in accordancewith the principles described herein by way of illustration and notlimitation; the specific examples are not intended to limit the scope ofthe present disclosure and the appended claims. Numerous modificationsand alternative compositions, methods, and systems may be devisedwithout departing from the spirit and scope of the present disclosure.

EXAMPLES

Unless otherwise indicated, materials in the experiments below may bepurchased from the Sigma-Aldrich Chemical Corporation (St. Louis Mo.) orFluka Chemical Corporation (Milwaukee Wis.). Parts and percentagesdisclosed herein are by weight to volume unless otherwise indicated.

DEFINITIONS

mg=milligram

g=gram(s)

ng=nanogram(s)

mL=milliliter(s)

μL=microliter(s)

μmol=micromolar

° C.=degrees Centigrade

min=minute(s)

sec=second(s)

hr=hour(s)

w/v=weight to volume

TLC=thin layer chromatography

HPLC=high performance liquid chromatography

EtOAc=ethyl acetate

DMF=dimethylformamide

DMSO=dimethylsulfoxide

MeOP=1-methoxy-2-propanol

MES=2-(N-morpholino)ethanesulfonic acid

DI=distilled

UPA=Ultra Particle Analyzer

LOCI=luminescent oxygen channeling immunoassay

BSA=bovine serum albumin

BGG=bovine gamma globulin

mIgG=mouse immunoglobulin

MS=mass spectrometry

Preparation of EPRM-EDA Beads

EPRM beads (2000 mg, 20.0 mL) are added to a 40-mL vial. The EPRM beadsare prepared by a procedure similar to that described in U.S. Pat. No.7,179,660 and the chemiluminescent compound is 2-(4-(N,N,di-tetradecyl)-anilino-3-phenyl thioxene with europium chelate. EDA (800mg, 890 μL) is combined with 10 mL MES pH 6 buffer (the “Buffer”) andabout 4.2 mL 6N HCl. The pH of the mixture is, or is adjusted to be,about 6.9. The EDA solution is added to the EPRM beads with vortexingand the mixture is rocked at room temperature for 15 minutes. Sodiumcyanoborohydride (400 mg) is combined in a 15 mL vial with 10 mL DIwater and the combination is added to the bead mixture from above. Themixture is shaken at 37° C. for 18-20 hours. The beads are transferredto six 40 mL centrifuge tubes. MES buffer is added to bring the volumeto 35 mL and the mixture is centrifuged at 19,000 rpm for 30 min. Thesupernatant is decanted and the beads are re-suspended in 2 mL of theBuffer with a stir-rod and additional Buffer is added to 35 mL. Themixture is sonicated at 18 Watts power for 30 sec, using ice to keep themixture cold. The wash/sonication step is performed 4 times to removeall activation chemical. After the last MES Buffer centrifugation, 2 mLof the Buffer containing 5% MeOP and 0.1% Tween® 20 (the “secondBuffer”) is added to the tubes for the re-suspension step. Additionalsecond buffer is added to 35 mL before sonication. The bead suspensionis centrifuged at 19,000 rpm for 30 min. The supernatant is discarded.The final sonication used 12 mL of the second Buffer in each tube togive a 25 mg/mL dilution. Particle size is 277 nm as determined on a UPAinstrument.

The EPRM chemibead is prepared in a manner similar to the methoddescribed in U.S. Pat. No. 6,153,442 and U.S. Patent ApplicationPublication No. 20050118727A, the relevant disclosures of which areincorporated herein by reference. The EPRM chemibead comprises anaminodextran inner layer and a dextran aldehyde outer layer having freealdehyde functionalities. See, for example, U.S. Pat. Nos. 5,929,049,7,179,660 and 7,172,906, the relevant disclosures of which areincorporated herein by reference. The reaction is carried out at atemperature of about 0 to about 40° C. for a period of about 16 to about64 hours at a pH of about 5.5 to about 7.0, or about 6, in a bufferedaqueous medium employing a suitable buffer such as, for example, MES.The reaction is quenched by addition of a suitable quenching agent suchas, for example, carboxymethoxyamine hemihydrochloride (CMO), andsubsequent washing of the particles.

Aldehyde groups on the outer dextran aldehyde layer are reacted withethylene diamine under reductive amination conditions to form reagentEPRM-EDA having pendant moieties comprising an ethylene chain and aterminal amine group. The reductive amination conditions include the useof a reducing agent such as, for example, a metal hydride. The reactionis carried out in an aqueous medium at a temperature during the reactionof about 20° C. to about 100° C. for a period of about 1 hour to about48 hours.

Synthesis of 25-OH Vitamin D₃ 3-carbamate (25-OH Vitamin D₂ 3-carbamate)

Referring to FIG. 1, a mixture of 22 mg (55 μmol) 25-OH VD₃ (I)purchased from ChemReagents.com, Sugarland Tex., 100 mg (420 μmol)disuccinimidyl carbonate (DSC), 100 μL triethylamine in 1 mL anhydrousacetonitrile in a 5-ml flask (covered with foil) was stirred at roomtemperature for 18 hr under nitrogen to prepare activated 25-OH VD₃(II). TLC (EtOAc:Hexane=2:1) showed no starting material left. Asuspension was prepared by adding 150 mg of carboxymethoxylaminehemihydrochloride (CMO), 0.3 ml triethylamine and 1 ml DMF to a 10 mlflask. A solution containing activated 25-OH VD₃ was added dropwise tothe CMO suspension with stirring, which was continued for another 18 hr.Vacuum was applied to remove the solvents as much as possible (theheating bath temperature should not be over 50° C.). EtOAc (25 ml) wasadded to the residue, which was washed three times with 2 ml brine. Theorganic phase was dried with anhydrous Na₂SO₄ and was filtered; solventwas removed using rotavap. Crude product (42 mg) was obtained afterdrying and was purified by HPLC. Pure product (III) (p=1) (24 mg) wasobtained after being dried under high vacuum. The product was dissolvedinto 1.2 ml anhydrous DMSO. Aliquots were transferred into vials, whichwere kept at −70° C.

A method similar to that described above was employed to prepare 25-OHVitamin D₂ 3-carbamate using 25-OH VD₂.

Synthesis of 25-OH Vitamin D₃ 3-succinate (25-OH Vitamin D₂ 3-succinate)

A mixture of 14.2 mg (35.4 μmol) 25-OH VD₃, 8.2 mg (90 μmol) succinicanhydride, 8.2 mg imidazole in 1 mL anhydrous dichloromethane in a 5-mlflask was stirred at room temperature for 24 hr under nitrogen. TLC(EtOAc:Hexane=2:1) showed no starting material left. Solvent was removedunder vacuum using a heating bath under 50° C. EtOAc was added to theresidue, which was then washed three times with 3×2 ml brine. Theorganic phase was removed with anhydrous Na₂SO₄ and was filtered.Solvent was removed using a rotavap and the resulting material wassubjected to drying under high vacuum to give 18 mg of crude product,which was confirmed to be 25-OH Vitamin D3 3-succinate by MS.

A method similar to that described above was employed to prepare 25-OHVitamin D₂ 3-succinate using 25-OH VD₂.

Coupling of EPRM-EDA and 25-OH Vitamin D₃ 3-Carbamate to Give ParticleReagent VI

Referring to FIGS. 1 and 2, carbamate III (10 μL of aliquot in DMSOprepared as described above) (0.2 mg) was added to a 2-mL vial labeled3323-064-1. EDAC (6.8 mg) and SNHS (9.4 mg) plus 2.27 mL dry DMSO (3mg/mL) were added to a 5-mL vial labeled EDAC/SNHS. The EDAC/SNHSsolution (190 μl) was added to vial 3323-064-1 (1 mg/mL) to prepareactivated 25-OH vitamin D₃ 3-carbamate IV. The mixture was allowed torotate at room temperature for 18 hr. A 0.4 mL aliquot of a 16% GAFAC®surfactant solution (GAF Corporation, Wayne N.J.) (0.15%) was diluted to1.6% with 3.6 mL DI water.

Vitamin D₃ (8.5 mg) and 850 μl, DMSO (10 mg/mL) were combined. To a10-mL round bottom flask (labeled 3323-064B) equipped with a stir-barwas added 2.0 mL (200MG) EPRM-EDA followed by 400 μL (4 mg) of theVitamin D₃ solution from above. The mixture stirred overnight at roomtemperature.

To a 10-mL round bottom flask (designated 3323-64A) equipped with astir-bar was added 2.0 mL (200 mg) EPRM-EDA (prepared as describedabove) followed by 260 μL 1.6% GAFAC® surfactant solution (0.15%) withmoderate stirring. To a small test tube was added 504 μl, anhydrous DMSOfollowed by 60 μl, (0.06 mg) 3323-064-1 activated Vitamin D₃-3-carbamateIV prepared as described above; and the mixture was added to theEPRM-EDA bead mixture. The total DMSO content of the 3323-64A the beadsuspension was 20%.

To the 10-mL round bottom flask (designated 3323-064B) was added 260 μL1.6% GAFAC® surfactant solution (0.15%) with moderate stirring. To asmall test tube was added 104 μl, anhydrous DMSO followed by 60 μl,(0.06 mg) 3323-064-1 activated vitamin D₃-3-carbamate IV. TheDMSO/activated vitamin D₃-3-carbamate IV was added to the bead mixture.The combined reactants (Vitamin D₃/DMSO) plus added DMSO gave a 20% DMSOcontent to the 3323-064B bead suspension. The two reaction vessels wereallowed to stir overnight at room temperature. Then, the beads werewashed by means of diafiltration.

Each bead lot was taken up to 20 mL working volume with 10% MeOP/1%GAFAC®/MES pH6 buffer. The mixture was diafiltered with 5 volumes of thebuffer and then sonicated with a probe sonicator at 18-21 Watts usingice to keep the mixture cold. The diafiltration/sonication continuedthrough 50 volumes with effluent samples being taken at 35, 40, 45 and50 volumes. The buffer was changed to LOCI Hapten Wash Buffer (50 mMHEPES, 300 mM NaCl, 1 mM EDTA, 0.01% neomycin sulfate, 0.1% TRITON® 405Xand 0.15% PROCLIN® 300, pH 7.2) with 10 volumes being used. The mixturewas reduced to about 7 mL and a UPA performed. Particle sizes were3323-064A=289 nm and 3323-064B=298 nm. Percent solids were determinedand both bead lots were brought up to 10 mg/mL with LOCI Hapten WashBuffer pH7.2. Yield was as follows: 3323-064A=160.4 mg and3323-064B=183.5 mg.

Coupling of EPRM-EDA and 25-OH Vitamin D₂ 3-Carbamate to Give ParticleReagent VI

In a manner similar to the described above for the preparation ofparticle reagent VI, 25-OH Vitamin D₂ 3-Carbamate was coupled toEPRM-EDA to give a similar particle reagent.

Coupling of EPRM-EDA and 25-OH Vitamin D₃ 3-succinate

25-OH Vitamin D₃ 3-succinate (66 μL of aliquot in DMSO prepared asdescribed above) (2 mg) was added to a 2-mL vial labeled 3323-026-1.EDAC (20 mg) and NHS (20 mg) plus 0.8 mL dry DMSO (25 mg/mL) were addedto a 5-mL vial labeled EDAC/NHS. The EDAC/NHS solution (180 μL) wasadded to vial 3323-026-1 (8.1 mg/mL) to prepare activated vitaminD₃-3-succinate. The mixture was allowed to rotate at room temperaturefor 18 hr. To a 2-mL centrifuge tube 3323-026A was added 0.5 mL (50 mg)EPRM-EDA followed by 1.4 ml MES pH6 followed by (119 μL) (0.96 mg) ofactivated vitamin D₃-3-succinate (with vortexing during addition). Theaddition gave a 5.9% DMSO content to the beads.

To a 2-mL centrifuge tube labeled 3323-026B was added 0.5 mL (50 mg)EPRM-EDA followed by 1.4 ml MES pH6 followed by 69 μL DMSO plus 30 μL(0.24 mg) of activated Vitamin D₃-3-Succinate (with vortexing duringaddition). The addition plus the extra DMSO gave a 5% DMSO content tothe beads.

Mixtures were transferred to 40-mL centrifuge tubes. MES pH6 Buffercontaining 10% MeOP and 1% GAFAC® was added to bring the volume in thetubes to 35 mL. Tubes were centrifuged at 18,500 rpm for 30 minutes.Supernatant was decanted.

Beads were re-suspended in 1 mL MES Buffer mixture with a stir-rod. MoreMES Buffer mixture was added to bring tubes to 35 mL. Using ice to keeptubes cold, the tubes were sonicated (probe sonicator) at 18-21 Wattspower for 60 seconds. The beads were centrifuged as above with a totalof four MES-MeOP-GAFAC® buffer washes being performed. After the lastwash, the beads were resuspended in 1 mL Hapten Wash Buffer instead ofthe MES Buffer mixture as above with two more washes being performed.The beads were resuspended in sufficient Hapten Wash Buffer to give a 15mg/mL suspension. Beads were sonicated at 50% power (cup sonicator) for60 seconds. Particle size was determined on a UPA instrument. Particlesize for 3323-26A was 290 nm; particle size for 3323-26B was 275 nm.Yield was as follows: 3323-26A=33.2 mg and 3323-26B=32.3 mg.

Assay for Vitamin D Analyte

Assays were carried out on a DIMENSION® VISTA® analyzer (SiemensHealthcare Diagnostics Inc., Deerfield, Ill.) following the protocol fora LOCI assay and using sample solutions containing varying amounts of25-hydroxyvitamin D3. In this example, the assay uses, as achemiluminescent reagent, a label particle-conjugate (“chemibead(s)”) inaccordance with the principles described herein where the label of theparticle-conjugate is a chemiluminescent compound contained in a latexparticle. Samples were reacted first with a biotinylated antibodyagainst 25-hydroxyvitamin D and then with chemibeads. The chemibeadsbind to the fraction of the monoclonal antibody binding sites that isnot occupied by analyte from the sample. Subsequently, streptavidincoupled sensitizer beads are added to the reaction mixture. This leadsto the formation of chemibead/sensibead pairs whose concentration isinversely related to the concentration of 25-hydroxyvitamin D3. Uponillumination at 680 nm, the sensitizer beads generate singlet oxygenwhich diffuses into the chemibeads which are paired with sensibeads,reacts with the olefinic dye and triggers a chemiluminescent signal atapproximately 612 nm which is inversely related to the analyteconcentration.

The streptavidin-sensitizer bead (“sensibead(s)”) is prepared using amethod analogous to that described in U.S. Pat. Nos. 6,153,442,7,022,529, 7,229,842 and U.S. Patent Application Publication No.20050118727A. The photosensitizer wasbis-(trihexyl)-silicon-t-butyl-phthalocyanine The concentration ofsensibead reagent was 200 μg/mL in HEPES buffer, pH 8.0 containing 150mM NaCl. The EPRM-EDA-25-OH Vitamin D₃ particle reagent VI prepared asdescribed above was employed as a “chemibead reagent” at a concentrationof 200 μg/mL in HEPES buffer, pH 7.2, containing 150 mM NaCl and 0.1%detergent.

The biotinylated antibody reagent is prepared using monoclonal antibodyspecific for 25-hydroxyvitamin D (sheep monoclonal from Bioventix,Farnham, Surrey, UK) and biotinylating amine groups of the antibody byreaction with NHS-PEO4-biotin (Pierce Chemical Company, Rockford Ill.)in a manner similar to that described in U.S. Patent ApplicationPublication No. 2009/0258435A1, the relevant portions of which areincorporated herein by reference. The biotinylated antibody reagent isprepared in HEPES buffer pH 7.2 containing 150 mM NaCl and 16 mg/mL BSA,1 mg/mL mIgG and 2 mg/mL BGG where the concentration of biotinylatedantibody reagent is 1000 ng/mL.

The assays were also carried out as above with the exception thatEPRM-EDA-25-OH vitamin D₃ succinate particle reagent was employed inplace of EPRM-EDA-25-OH Vitamin D₃ particle reagent VI.

At time t=zero sec, 20 μL biotinylated antibody reagent and 20 μL waterwere added to a reaction vessel. Sample, 12 μL, was added 21.6 secondslater, followed by 8 μL water. At t=414.0 seconds, 40 μL chemibeadreagent was added followed by 20 mL of water. Sensibead reagent was thendispensed at 457.2 seconds. Measurements were taken 601.2 seconds afterinitiation of the reaction sequence.

The results are summarized in Table 1 below.

TABLE 1 kcounts 25-OH-D₃-3- 25-OH-D₃-3- carbamate succinate Vitamin D(ng/mL) 9.6 mg/g 4.8 mg/g  9.6 mg/g 4.8 mg/g  0 1118 812 414 653 2 1135818 417 662 5 1128 813 410 654 23 1039 744 376 594 216 651 488 237 387528 418 337 159 269

As can be seen from Table 1, the 25-OH-D₃-3-carbamate chemibead reagentin accordance with the principles described herein gave consistentlyhigher signal counts compared to the 25-OH-D₃-3-succinate chemibeadreagent.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

It should be understood that the above-described examples are merelyillustrative of some of the many specific examples that represent theprinciples described herein. Clearly, those skilled in the art canreadily devise numerous other arrangements without departing from thescope as defined by the following claims.

What is claimed is:
 1. A compound of the formula:

wherein: Z is an alkyl, alkenyl or alkynyl group having 1 to 10 carbonatoms, which may be unsubstituted or one or more of which may besubstituted by one or more of hydroxy, lower alkoxy, oxo, and oxime; Ris —O-succinimidyl, —NH—O-(A)_(k)-R₂, wherein R₂ is a member of a signalproducing system, —(CH₂)nCOOH, —(CH₂)mCOONHS (NHS isN-hydroxysuccinimide), a small molecule, a binding partner for a smallmolecule, or a support; A is a linking group; k is 0 or 1; n is aninteger of 1 to 5 and m is an integer of 1 to 5; and R⁴ is H, OH orlower alkoxy.
 2. The compound according to claim 1 wherein Z is abranched alkyl group or a branched alkenyl group having 4 to 10 carbonatoms wherein a terminal carbon atom of the branched alkyl group or thebranched alkenyl group comprises a hydroxyl.
 3. The compound accordingto claim 1 wherein R² is a member of a signal producing system selectedfrom the group consisting of fluorescent compounds, chemiluminescentcompounds, sensitizers, enzymes and radiolabels.
 4. A compound of theformula:

or a compound of the formula:

wherein: R′ is —O-succinimidyl or —NH—O-(A)_(k)-R², wherein R² is amember of a signal producing system, —(CH₂)_(n)COOH, —(CH₂)_(m)COONHS(NHS is N-hydroxysuccinimide), a small molecule, a binding partner for asmall molecule, or a support; A is a linking group; k is 0 or 1; n is aninteger of 1 to 5 and m is an integer of 1 to 5; R¹ is H, OH, orprotected OH; and R⁴ is H, OH or lower alkoxy.
 5. The compound accordingto claim 4 wherein R² is a member of a signal producing system selectedfrom the group consisting of fluorescent compounds, chemiluminescentcompounds, sensitizers, enzymes and radiolabels.
 6. The compoundaccording to claim 4 wherein R² is a member of a signal producing systemthat comprises a particle.
 7. The compound according to claim 6 whereinthe particle is selected from the group consisting of fluorescentparticles, chemiluminescent particles, sensitizer particles and magneticparticles.
 8. A method of determining in a sample the presence and/oramount of vitamin D, the method comprising: (a) providing in combinationin a medium: (i) the sample, (ii) the compound according to claim 5, and(iii) a specific binding member for vitamin D; (b) subjecting thecombination to conditions for binding of the compound of claim 1 to thespecific binding member to form a complex, and (c) measuring the amountof the complex, the amount of the complex being related to the presenceand/or amount of vitamin D in the sample.
 9. A compound of the formula:

or a compound of the formula:

wherein: L is selected from the group consisting of chemiluminescentparticles and sensitizer particles and R^(1′) is OH and A′ is a linkinggroup.
 10. The compound according to claim 9 wherein L is achemiluminescent particle.
 11. The compound according to claim 9 havingthe formula:

or the formula:

wherein: p is an integer from 1 to 5, q is an integer from 1 to 5 and ris an integer from 1 to
 5. 12. The compound according to claim 11wherein L is a chemiluminescent particle.
 13. An assay for vitamin Dcomprising employing as one reagent in the assay the compound accordingto claim
 9. 14. A method of determining in a sample the presence and/oramount of vitamin D, the method comprising: (a) providing in combinationin a medium: (i) the sample, (ii) the compound according to claim 9, and(iii) a specific binding member for vitamin D; (b) subjecting thecombination to conditions for binding of the compound of claim 1 to thespecific binding member to form a complex, and (c) measuring the amountof the complex, the amount of the complex being related to the presenceand/or amount of vitamin D in the sample.
 15. A method of determining ina sample the presence and/or amount of vitamin D, the method comprising:(a) providing in combination in a medium: (i) the sample, (ii) acompound of the formula:

or a compound of the formula:

wherein: R″ is —NH—O-A″—R^(2′), wherein R^(2′) is a member of a signalproducing system and A″ is a linking group, and R^(1″) is H or OH; and(iii) a specific binding member for vitamin D; (b) subjecting thecombination to conditions for binding of the compound of to the specificbinding member to form a complex, and (c) measuring the amount of thecomplex, the amount of the complex being related to the presence and/oramount of vitamin D in the sample.
 16. The method according to claim 15wherein A″ is —(CH₂)_(p)—C(O)—(W)_(b)—(CH₂)_(q)—(X)_(c)—(CH₂)_(r)—wherein W and X are each independently —NH— or —O—, b and c are eachindependently 0 or 1, p is an integer of 1 to 5, q is an integer of 1 to5, and r is an integer of 1 to
 5. 17. The method according to claim 15wherein the member of the signal producing system is selected from thegroup consisting of sensitizers and chemiluminescent compounds.
 18. Themethod according to claim 17 wherein the member of the signal producingsystem is associated with a particle.
 19. The method according to claim15 wherein the member of the signal producing system is aphotosensitizer and the combination further comprises a chemiluminescentreagent.
 20. The method according to claim 15 wherein the member of thesignal producing system is a chemiluminescent compound and thecombination further comprises a photosensitizer reagent.